This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding geranylgeranyl pyrophosphate synthase or geranylgeranyl pyrophosphate synthase-related protein in plants and seeds.
Geranylgeranyl pyrophosphate (GGPP) synthase, also known as geranylgeranyl-diphosphate synthase, farnesyl transferase and geranylgeranyl synthetase is a key enzyme in plant terpenoid biosynthesis. The final product, GGPP, is the key precursor of several holoterpenoids such as carotenoids and meroterpenoids. One fate of GGPP is conversion to phytoene by phytoene synthase, the first committed step in carotenoid biosynthesis. Although not specific to carotenoid biosynthesis, GGPP synthase may be important in determining the total catorenoid content of a specific tissue. Expression of the GGPP synthase gene is strongly induced during the chloroplast to chromoplast transition which occurs in ripening peppers which have a high carotenoid content (Kuntz, M., et al. (1992) Plant J. 2:25-34).
GGPP also serves as precursor in the formation of defense-related substances like the phytoalexin casbene in castor bean and the diterpene phorbol which acts as a toxin against herbivores. GGPP is also a precursor of the important phytohormone gibberellin which regulates a variety of physiological processes that include initiation of seed germination, stimulation of stem elongation, stimulation of flowering/bolting and regulation of leaf/fruit senescence.
In animal systems, the importance of the enzyme GGPP synthase is demonstrated by the lethality of nonsense mutations in the locus that encodes the enzyme in Drosophila (Lai et al. (1998) Genetics 149:1051-1061). In plant systems, GGPP serves as precursor to many important metabolites that the enzyme responsible for its synthesis, GGPP synthase, appears to be an attractive target for herbicide discovery and design.
At least 6 different GGPP synthases have been identified in Arabidopsis thaliana. Beside differences in the amino acid sequence of the proteins and the nucleotide sequence of their genes, GGPP synthases accumulate in different subcellular compartments (Zhu, X. F., et al. (1997) Plant Mol. Biol. 35:331-341).
Manipulation of the corn gene in endosperm could result in increased xanthophyll content, which has value as coloring agent in poultry feed.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 35 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a corn geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:10. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:6, and a wheat geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:42. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 50 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a corn geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:2. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 100 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:8, a rice geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:12, a wheat geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:28, a wheat geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:30, a rice geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:32, a rice geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:34, a soybean geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:36, a wheat geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:40 and a wheat geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:44. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 100 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:4, a rice geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:14, and a soybean geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:20. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 150 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a soybean geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:24. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 150 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a soybean geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:22. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 200 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a soybean geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:38. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 200 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a soybean geranylgeranyl pyrophosphate synthase polypeptide of SEQ ID NO:18. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, and 43 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 35 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:10. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 50 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 42. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 50 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:2. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 100 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:8, 12, 28, 30, 32, 34, 36, 40, and 44. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 100 amino acids comprising at least 85% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:4, 14, and 20. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 100 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:16 and 26. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 150 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:24. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 150 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:22. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 200 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:38. The present invention also relates to a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide of at least 200 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:18.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide in a host cell, preferably a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a host cell;
measuring the level a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide in the host cell containing the isolated polynucleotide; and
comparing the level of a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide in the host cell containing the isolated polynucleotide with the level of a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide in a host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide gene, preferably a plant geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein in the transformed host cell; (c) optionally purifying the geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein expressed by the transformed host cell; (d) treating the geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein with a compound to be tested; and (e) comparing the activity of the geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein that has been treated with a test compound to the activity of an untreated geranylgeranyl pyrophosphate synthase or a geranylgeranyl pyrophosphate synthase-related protein, thereby selecting compounds with potential for inhibitory activity.